Project Summary Cancer, the uncontrolled division of abnormal cells, is the second leading cause of death in the United States, taking the lives of nearly 600,000 Americans in 2015. In particular, metastatic spread of tumor cells is responsible for the vast majority of cancer related deaths. As such, understanding the changes that cancer cells face during the metastatic cascade is key to tackling cancer morbidity and mortality. Unfortunately, our understanding of signaling pathways and transcriptomic changes contributing to metastasis is still limited. This has been due, in part, to the lack of human specimens from early metastatic intermediates serving as good experimental models and to the lack of unbiased, genome-wide screening approaches with which to probe these models. However, recent work from my group has led to the establishment of ex vivo cultures of breast cancer circulating tumor cells (CTCs), which more accurately represent the cells directly responsible for initiation of metastases. Additionally, several groups have recently developed genome-wide CRISPR screening approaches, including knockout, activation, and inactivation libraries. The merging of these two technologies will allow for the identification of novel transcriptomic changes contributing to progression through the metastatic cascade. With this grant, I propose to use novel genome-wide CRISPR activation technology in vivo to identify genes which, when modulated, enhance the ability of CTCs to progress through the metastatic cascade. Aim 1 will use a genome-wide CRISPR activation screening library in two circulating tumor cell cultures to identify genes capable of enhancing metastatic potential in vivo. CTCs carrying the screening library will be injected into mouse tail veins, and after two months, mouse lungs will be analyzed via next-generation sequencing to identify single guide RNAs (sgRNAs) and corresponding genes enriched in the lungs. Aim 2 will clinically and functionally validate gene hits. Clinical databases, as well as an in-house database of patient-derived single CTCs and CTC clusters, will be analyzed to provide clinical validation. sgRNAs against gene hits will be tested in competition in vivo, to provide both functional validation and ranking of magnitude of enhanced metastatic potential. Aim 3 will determine the mechanistic underpinnings allowing gene hits to enhance metastatic potential. Loss-of-function experiments will be performed to identify whether gene hits are necessary or sufficient for metastasis. RNA- sequencing and proteomic analyses will be performed to define downstream targets and pathways, allowing for identification of cellular processes which may be contributing to the pro-metastatic phenotype. Finally, if small molecules are available against gene hits, these small molecules will be tested for ability to inhibit metastatic potential of CTCs in vivo. By leveraging circulating tumor cell cultures, novel genome-wide CRISPR activation libraries, and next-generation sequencing technologies, this proposal will help identify transcriptomic changes that play a role in cancer growth and metastasis, as well as suggest novel therapeutic opportunities.